Solution structure and dynamics of polyubiquitin chains

本研究旨在研究真核生物中普遍存在的蛋白水解信号——多聚素（polybiquitin, polyb）链的溶液结构和动力学特性。泛素-蛋白酶体蛋白水解途径是影响多种重要细胞事件的短寿命蛋白周转的主要调控机制。了解polyb链如何被26S蛋白酶体识别是理解调控机制的核心。尽管有重要证据表明，对不同聚脲链的特异性识别是基于构象决定因素的，但迄今为止，对这些分子在溶液中的构象性质还没有明确的认识。获得这样的信息是绝对必要的，以便发展对不同链如何能够作为特定信号的分子理解。我们将使用核磁共振方法来确定在溶液中游离并与蛋白酶体的肽模拟物结合的四聚氨基乙基链的三维构象。这将使用来自自旋弛豫和残余偶极耦合测量的远程定向约束来执行。这些新的核磁共振方法将与基于NOES的传统方法以及基于化学位移微扰和溶剂可及性测量的域间或分子间界面的映射相结合。在这些研究中，我们将使用体外偶联反应，使用由细菌表达的Ub单体（未标记和均匀标记）组装的节段同位素标记链。我们将测量双Ub和四Ub的蛋白质动力学，并与单体Ub进行比较，以确定结构域间运动的贡献，并表征polyb链的构象灵活性。

This proposal is to characterize the solution structure and dynamics of polyubiquitin (polyUb) chains, a universal proteolytic signal in eukaryotes. The ubiquitin-proteasome proteolytic pathway is the principal regulatory mechanism for the turnover of short-lived proteins that influences a variety of vital cellular events. Understanding how polyUb chains are recognized by the 26S proteasome is central to understanding of the mechanisms of regulation. Although significant evidence suggests that the specific recognition of different polyUb chains is based on conformational determinants, as yet there is no clear view of the conformational properties of these molecules in solution. Obtaining such information is absolutely necessary in order to develop a molecular understanding of how different chains are able to act as specific signals. We will use NMR approaches to determine the three-dimensional conformation of the tetra-Ub chain free in solution and bound to a peptide mimic of the proteasome. This will be performed using long-range, orientational constraints derived from spin-relaxation and residual dipolar couplings measurements. These novel NMR approaches will be used in combination with the conventional methods based on NOES and mapping of the interdomain or intermolecular interface based on chemical shift perturbations and solvent accessibility measurements. For these studies, we will use segmentally isotope-labeled chains assembled from bacterially-expressed Ub monomers (unlabeled and uniformly labeled) using in vitro conjugation reaction. We will measure protein dynamics in the dual Ub and tetra-Ub, in comparison with that of the monomeric Ub, in order to determine the contribution from interdomain motions and characterize the conformational flexibility of polyUb chains.